Treatment of Cardiovascular Disorders Using the Cell Differentiation Signaling Protein Nell1

ABSTRACT

It has been identified in accordance with the present invention that Nell1 is essential for normal cardiovascular development by promoting proper formation of the heart and blood vessels. The present invention therefore provides therapeutic methods for treating cardiovascular disorders by employing a Nell1 protein or nucleic acid molecule.

CROSS REFERENCE TO RELATED APPLICATIONS

This application asserts the priority of U.S. provisional applicationSer. No. 60/995,854 filed Sep. 28, 2007, and U.S. provisionalapplication Ser. No. 61/079,446, filed Jul. 10, 2008, the contents ofwhich are incorporated herein by reference.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

This invention was made with Government support under Contract No.DE-ACO5-000R22725 between the United States Department of Energy andUT-Battelle, LLC. The Government has certain rights in this invention.

FIELD OF THE INVENTION

The present invention relates in general to therapeutic methods fortreating cardiovascular disorders. More specifically, the presentinvention relates to therapeutic treatments of cardiovascular disordersby employing the cell differentiation signaling protein Nell 1, as wellas functional derivatives thereof.

BACKGROUND OF THE INVENTION

Despite many available methods of treatment, cardiovascular disease isone the major causes of death each year in the U.S. Thus, there is stilla need for more effective agents to prevent and treat cardiac tissueinjury, especially cardiac tissue injury resulting fromischemia/reperfusion.

The Nell1 gene codes for a secreted trimeric protein that stimulatesbone and cartilage precursor cells (osteoblasts and chondrocytes) todifferentiate into mature bone and cartilage tissue (Zhang et al., 2002;Desai et al., 2006). Nell-1 is a protein kinase C (PKC) β-bindingprotein. The Nell1 cDNA and amino acid sequences from a variety ofmammalian species, including human, rat and mouse, have been reported.

Overexpression of Nell1 has been reported to cause premature fusion ofthe growing cranial bone fronts, resulting in craniosynostosis in humansand transgenic mice carrying a rat Nell 1 transgene. A Nell1 knock-outmouse was also shown to exhibit several bone- and cartilage-relateddefects. There has been no characterization, however, of the impact ofNell 1, if any, on cardiovascular development.

SUMMARY OF THE INVENTION

It has been identified in accordance with the present invention thatNell1 is essential for normal cardiovascular development by promotingproper formation of the heart and blood vessels. The present inventiontherefore provides therapeutic methods for treating cardiovasculardisorders by employing a Nell1 protein, functional derivatives thereofor nucleic acid molecule.

Cardiovascular disorders or conditions contemplated by the presentinvention are diseases that involve the heart or blood vessels (arteriesand veins), including in particular myocardial infarction (or “MI”). Bytreating a cardiovascular disorder or condition with the presentmethodology, the disorder is prevented or is delayed; or alternatively,its progression is slowed down, the extent of the injury is reduced, andthe recovery is accelerated.

In one embodiment, the present invention provides a method of treating acardiovascular disorder by administering a Nell1 protein or functionalderivatives thereof to a subject in need of the treatment. Nell 1proteins suitable for use in the present method include wild type Nell 1proteins from any mammalian species, as well as functional derivativesthereof. Nell1 proteins, as well as functional derivatives thereof, canbe recombinantly produced or purified from a mammalian body or tissue.

In another embodiment, the present invention provides a method oftreating a cardiovascular disorder by administering a nucleic acidmolecule encoding a Nell1 protein to a subject in need of the treatment.The nucleic acid molecule can be provided in an expression vector,including viral vectors and non-viral vectors, suitable for effectingthe expression of the Nell1 protein in the targeted tissue or cells.

In accordance with the present invention, a Nell1 protein, functionalderivatives thereof, or nucleic acid molecule can be combined with anappropriate pharmaceutically acceptable carrier for administration.Administration can be conducted in any practical and convenient manner,including by ingestion, injection or implantation, for example.

In a specific embodiment, a Nell1 protein, functional derivativesthereof, or Nell1-encoding nucleic acid molecule is used in combinationwith cell-based therapy for the repair and regeneration of damagedcardiac muscles and blood vessels. For example, a Nell1 protein,functional derivatives thereof, or Nell1-encoding nucleic acid moleculecan be administered together with cardiomyocytes for repopulation ofcells in the injured site. Alternatively, a Nell 1 protein, functionalderivatives thereof, or Nell 1-encoding nucleic acid molecule can beadministered together with stem cells isolated from adult bone marrowfor regeneration of damaged cardiac muscles and blood vessels.

BRIEF DESCRIPTION OF THE DRAWINGS

The file of this patent application contains drawings executed in color.

FIGS. 1A-1B show the cardiovascular defects in mice without Nell1function (Nell1^(6R) mutation). Homozygote fetuses at E18 days ofgestation (Top) show decreased blood circulation (arrows) and unexpandedlungs compared to heterozygotes (bottom) and wild type animals (notshown). Fetuses were unable to breathe after birth or after caesareanrecovery.

FIGS. 2A-2B (in color) demonstrate that Nell 1 protein is required forblood vessel formation and establishment of a complex vascular network.The loss of Nell 1 function resulted in a significant reduction of thenumber of blood vessels and extensive branching of the vasculature inNell1^(6R) mutants (FIG. 2B) compared to (FIG. 2A) normal fetuses. Thedecrease in blood vessel formation was observed throughout the fetalbody.

FIGS. 3A-3B illustrate severe cardiovascular defects and neonatallethality associated with the complete loss of Nell 1 function in themouse. The cardiovascular defects resulting from the complete loss ofNell 1 function in Nell1^(6R) was associated with decreased bloodcirculation into the heart muscles and predominance of increased numbersof immature cardiomyocytes. The dense packing of smaller cardiomyocytesin the mutant (FIG. 3B) was very apparent in the denser/darker stainingwith haematoxylin and eosin, compared to the wild type (FIG. 3A). Thesecardiovascular defects are evident in E18.5 day fetuses recovered bycaesarean.

FIGS. 4A-4C illustrate a strategy for treatment of heart muscle injuryafter MI in rodents using direct injection of stem cells or drugs to theborder zone.

FIG. 5 provides an alignment of the human (SEQ ID NO: 2) and murine (SEQID NO: 4) Nell1 proteins. The functional domains of the human Nell 1protein are found in the essentially same regions as those identified inthe murine Nell1 protein.

FIGS. 6A-6D. NELL1 Protein Treatment of Damaged Heart Tissue in Micewith Myocardial Infarction (MI). (6A) Untreated mouse hearts with MI dueto the loss of blood supply from a ligation of the left anteriordescending coronary artery had a readily visible creamy white lookingdamaged tissue on the surface of the heart (17 days post MI-induction).All Nell1 protein treated hearts had lesser amount of damaged tissue asillustrated in FIG. 7B to 7D. The damaged sections (outlined by bluelines) in controls were typically at least 50% while the treated heartshad barely visible (6B) to as high as 30% infarcts observed (6D).

FIGS. 7A-7F. Reduction of Damaged Heart Tissue Incurred From MyocardialInfarction in Nell1-treated Hearts. Longitudinal sections of normalhearts stained with either haematoxylin and eosin (7A) ormasson-trichome (7B) show intense staining of the heart muscle andreveals a very compact organization of the muscle tissues in the rightand left ventricles (ry and lv respectively), and the interventricularseptum separating the two ventricular chambers (IVS). After a myocardialinfarction event, the muscle tissues died due to a lack of oxygenatedblood supply and the deterioration of the muscle architecture wasevident by the large gaps in the tissue and the decreased intensity ofthe staining (7C; 17 days post-MI). Hearts with MI that were treatedwith the Nell1 protein had lesser damage in the heart tissue from thesurface to just before the middle of the heart (7D and 7E). In somehearts the improvement was manifested even deeper into the middlesection of the heart (7F).

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to therapeutic methods for treatingcardiovascular conditions or disorders by employing the celldifferentiation signaling protein Nell 1, as well as functionalderivatives thereof.

The present invention is based on the surprising discovery by theinventor that the Nell 1 protein is essential for normal cardiovasculardevelopment by promoting proper formation of the heart and bloodvessels. The inventor discovered that loss of Nell 1 resulted in severaltissue and organ changes typical of cardiac muscle injury, includingheart enlargement, tissue hypertrophy, decreased blood vessel formationand blood circulation. The inventor observed that microscopicexamination of Nell1-deficient hearts showed heart enlargement andcardiomyopathy, conditions associated with events of myocardialinfarction (“MI”). Although the basic vasculature system was observedduring embryo development even without a functional Nell 1, the amountand complexity (branched network) was significantly reduced in Nell1mutants. The therapeutic application of Nell 1 for heart muscleregeneration is therefore dependent not only on the protein's abilitiesto signal muscle cell maturation, but also in its capabilities tosupport the construction of the highly branched vasculature that isrequired to sustain new heart muscle formation and maintenance of heartfunction. The inventor also observed that microarray experimentsindicate that Nell1 is essential for the proper formation of heartextracellular matrix, main structural components of heart muscle, andproper functioning of genes for heart metabolism and contraction.

Accordingly, the present invention provides methods for treatingcardiovascular conditions or disorders by employing a Nell1 protein,functional derivatives thereof, or Nell1 nucleic acid molecules.

The term “condition,” as used herein, refers to a disease or ailment.The term “disorder,” as used herein, refers to a condition in whichthere is a disturbance of normal functioning. The term “cardiovascular,”as used herein, refers to the heart and/or blood vessels.

Accordingly, the term “cardiovascular condition” or “cardiovasculardisorder”, as used herein, refers to diseases or aliments that involvethe heart, blood vessels (e.g., arteries and veins). Generally, suchdiseases or aliments result in an abnormality in the cardiac structure,cardiac muscle, and/or cardiac function. The cardiovascular condition ordisorder can be acute or chronic.

The term “cardiovascular condition” or “cardiovascular disorder” can beused interchangeably throughout the specification. Examples of acardiovascular disease include aneurysms, angina, atherosclerosis,cardiomyopathy, congestive heart failure, coronary artery disease, andmyocardial infarction, among others. Further examples of cardiovascularconditions include, for instances, blood vessels that have beenrevascularized. Such patients generally have a stent placed in a bloodvessel (e.g., artery, etc.)

A cardiovascular condition especially suitable for being treated withthe method of the present invention is myocardial infarction (or “MI”).MI, also known as a “heart attack” or “heart failure”, is a medicalcondition that occurs when the blood supply to a part of the heart isinterrupted. MI is often caused by partial or complete occlusion of oneor more of the coronary arteries, usually due to rupture of anatherosclerotic plaque. The occlusion of the coronary artery results incardiac ischemia. The resulting ischemia or oxygen shortage causesdamage and potential death of heart tissue.

The term “treating” or “treatment” a disease, as used herein, refers topreventing or delaying the onset of the disease, or when the diseasedoes occur, retard the progression or ameliorate the symptoms of thedisease, reduce the extent of tissue injury or damage, or promoterecovery of the injured tissue and regeneration of new functional tissueor cells.

The subject suitable for receiving a treatment in accordance with thepresent invention includes any mammalian subject in need of thetreatment. In one embodiment, the subject is a human subject. A subjectin need of treatment includes both subjects who have been determined tohave a higher risk of developing a cardiovascular disease, and subjectswho have a cardiovascular disease, as well as subjects who have recentlyexperienced a cardiovascular event such as MI.

In one embodiment, the method of the present invention is achieved byadministration of a Nell1 protein to a subject in need of the treatment.

“A Nell1 protein” as used herein, includes wild type (i.e., naturallyoccurring) Nell1 proteins of any mammalian origin, such as human,murine, rat and the like. Preferred Nell1 proteins for use in thepresent invention include human Nell1 protein (SEQ ID NO: 2), murineNell1 protein (SEQ ID NO: 4), and rat Nell1 protein (SEQ ID NO: 6).

“A Nell1 protein” as used herein, also includes functional derivativesof a wild type Nell1 protein. A “functional derivative” refers to amodified Nell1 protein which has one or more amino acid substitutions,deletions or insertions as compared to a wild type Nell1 protein, andwhich retains substantially the activity of a wild type Nell1 protein.By “substantially” is meant at least 50%, at least 75%, or even at least85% of the activity of a wild type Nell1 protein. According to thepresent invention, in order for the functional derivative tosubstantially retain the activity or function of a wild type Nell1protein, the functional Nell1 derivative shares a sequence identity withthe wild type Nell1 protein of at least 75%, at least 85%, at least 95%or even 99%.

The structure of Nell1 proteins has been characterized (see, e.g.,Kuroda et al., 1999a; Kuroda et al., 1999b, Desai et al., 2006). Forexample, the murine Nell1 protein (SEQ ID NO: 4) is a protein of 810amino acids, having a secretion signal peptide (amino acids #1 to 16),an N-terminal TSP-like module (amino acids #29 to 213), a Laminin Gregion (amino acids #86 to 210), von Willebrand factor C domains (aminoacids #273 to 331 and 699 to 749), and a Ca²⁺-binding EGF-like domains(amino acids #549 to 586).

The secretion signal peptide domain of Nell1 protein is an amino acidsequence in the protein that is generally involved in transport of theprotein to cell organelles where it is processed for secretion outsidethe cell. The N-terminal TSP-like module is generally associated withheparin binding. von Willebrand factor C domains are generally involvedwith oligomerization of Nell1 Laminin G domains of Nell1 protein aregenerally involved in adherence of Nell1 protein to specific cell typesor other extracellular matrix proteins. The interaction of such domainswith their counterparts is generally associated with, for example,processes such as differentiation, adhesion, cell signaling or mediatingspecific cell-cell interactions in order to promote cell proliferationand differentiation. The Ca²⁺-binding EGF-like domains of Nell1 bindsprotein kinase C beta, which is typically involved in cell signalingpathways in growth and differentiation.

The amino acid sequence of Nell1 protein is very highly conserved,especially across mammalian species. For example, the murine Nell1protein shares about 93% sequence identity with the human Nell1 protein(SEQ ID NO: 2), which, in turn, shares about 90% sequence identity withthe rat Nell1 protein (SEQ ID NO: 4). Those skilled in the art can useany of the well-known molecular cloning techniques to generate Nell1derivatives having one or more amino acid substitutions, deletions orinsertions, taking into consideration the functional domains (e.g.,secretion signal peptide sequence, N-terminal TSP-like module, Laminin Gregion, von Willebrand factor C domain) of Nell1. See, for example,Current Protocols in Molecular Cloning (Ausubel et al., John Wiley &Sons, New York).

The minimum length of a Nell1 functional derivative is typically atleast about 10 amino acids residues in length, more typically at leastabout 20 amino acid residues in length, even more typically at leastabout 30 amino acid residues in length, and still more typically atleast about 40 amino acid residues in length. As stated above, wild typeNell1 protein is approximately about 810 amino acid residues in length.A Nell1 functional derivative can be at most about 810 amino acidresidues in length. For example, a Nell1 functional derivative can be atmost at most about 820, 805, 800, 790, 780, 750, 600, 650 600, 550, etc.amino acid residues in length.

Once a Nell1 protein derivative is made, such protein can be tested todetermine whether such derivative retains substantially the activity orfunction of a wild type Nell1 protein. For example, the ability of aNell1 derivative to bind PKC beta can be tested. Suitable assays forassessing the binding of Nell1 to PKC beta is described in e.g., Kurodaet al. (1999b). For example, protein-protein interaction can be analyzedby using the yeast two-hybrid system. Briefly, a modified Nell1 proteincan be fused with GAL4 activating domain and the regulatory domain ofPKC can be fused with the GAL4 DNA-binding domain. The activity ofbeta-galactosidase in yeast cells can be detected.

In addition, one can also test the ability of a Nell1 derivative tostimulate differentiation of precursor cells, which are in thecardiomyocyte lineage, towards mature cardiomyocytes. Maturity ofcardiomyocytes can be assessed cellularly (histology) and molecularly(expression of cardiac-specific proteins or extracellular matrixmaterials). Still further, a Nell1 derivative can be tested for itsability to drive osteoblast precursors to mature bone cells, bydetecting expression of late molecular bone markers or mineralization(i.e., calcium deposits). By comparing the activity of a Nell1derivative with that of a wild type Nell1 protein in one or more of theassays such as those described above, one should be able to determinewhether such derivative retains substantially the activity or functionof a wild type Nell1 protein.

A Nell1 protein or functional derivative thereof may be prepared bymethods that are well known in the art. One such method includesisolating or synthesizing DNA encoding the Nell 1 protein, and producingthe recombinant protein by expressing the DNA, optionally in arecombinant vector, in a suitable host cell, including bacterial, yeast,insect or mammalian cells. Such suitable methods for synthesizing DNAare, for example, described by Caruthers et al. 1985. Science230:281-285 and DNA Structure, Part A: Synthesis and Physical Analysisof DNA, Lilley, D. M. J. and Dahlberg, J. E. (Eds.), Methods Enzymol.,211, Academic Press, Inc., New York (1992).

Examples of suitable Nell1 nucleic acid sequences include SEQ ID NOs: 1,3, and 5. A Nell1 protein or functional derivative may also be madesynthetically, i.e. from individual amino acids, or semisynthetically,i.e. from oligopeptide units or a combination of oligopeptide units andindividual amino acids. Suitable methods for synthesizing proteins aredescribed by Stuart and Young in “Solid Phase Peptide Synthesis,” SecondEdition, Pierce Chemical Company (1984), Solid Phase Peptide Synthesis,Methods Enzymol., 289, Academic Press, Inc, New York (1997). Examples ofsuitable Nell1 amino acid sequences include SEQ ID NOs: 2, 4, 6, andderivatives thereof.

In another embodiment, the method of the present invention is achievedby administration of a nucleic acid molecule encoding a Nell1 protein orfunctional derivative to a subject in need of the treatment.

Suitable nucleic acid molecules for use in the present invention includenucleic acid molecules having a nucleotide sequence as set forth in SEQID NO: 1 (encoding the wild type human Nell1 protein), SEQ ID NO: 3(encoding the wild type murine Nell1 protein), and SEQ ID NO: 5(encoding the rat wild type Nell 1 protein), as well as degeneratesequences thereof. As used herein, the term “degenerate sequence” refersto a sequence formed by replacing one or more codons in the nucleotidesequence encoding wild type Nell1 protein with degenerate codes whichencode the same amino acid residue (e.g., GAU and GAC triplets eachencode the amino acid residue Asp).

In some embodiments, nucleic acid molecules for use in the methods ofthe present invention are provided in an expression vector. Expressionvectors for use in the present methods include any appropriate genetherapy vectors, such as nonviral (e.g., plasmid vectors), retroviral,adenoviral, herpes simplex viral, adeno-associated viral, polio virusesand vaccinia vectors. Examples of retroviral vectors include, but arenot limited to, Moloney murine leukemia virus (MoMuLV), Harvey murinesarcoma virus (HaMuSV), murine mammary tumor virus (MuMTV), and RousSarcoma Virus (RSV)-derived recombinant vectors. A Nell1-codingnucleotide sequence can be placed in an operable linkage to a promoterin the expression vector, wherein the promoter directs the expression ofthe Nell1 protein in the targeted tissue or cells, and includes both aconstitutive promoter and a tissue or cell-specific promoter.

A Nell 1 protein, functional derivative thereof or Nell 1-encodingnucleic acid molecule can be combined with a pharmaceutically acceptablecarrier and prepared in formulations suitable for administration to asubject by injections, implantations, inhalations, ingestions and thelike. Pharmaceutically acceptable carriers are described hereinabove andinclude oils, water, saline solutions, gel, lipids, liposomes, resins,porous matrices, binders, fillers and the like, or combinations thereof.The carrier can be liquid, semi-solid, e.g. pastes, or solid carriers.Except insofar as any conventional media, agent, diluent or carrier isdetrimental to the recipient or to the therapeutic effectiveness of theactive ingredients contained therein, its use the present invention isappropriate. Examples of carriers include oils, water, saline solutions,gel, lipids, liposomes, resins, porous matrices, binders, fillers,patches, and the like, or combinations thereof. The carrier can also bea controlled release matrix that allows optimum release of a Nell1protein or nucleic acid admixed therein.

The term “therapeutically effective amount” means the dose required toprevent or delay the onset, slow down the progression or ameliorate thesymptoms of the disorder. Precise dosages depend on the disease state orcondition being treated and other clinical factors, such as weight andcondition of the subject, the subject's response to the therapy, thetype of formulations and the route of administration. As a general rule,a suitable dose of a Nell 1 composition (i.e., including a Nell1 proteinor nucleic acid) for the administration to adult humans ranges fromabout 0.001 mg to about 20 mg per kilogram of body weight. In someembodiments, a suitable dose of a Nell1 composition for theadministration to adult humans is in the range of about 0.01 mg to about5 mg per kilogram of body weight. However, the precise dosage to betherapeutically effective and non-detrimental can be determined by thoseskilled in the art.

A Nell1 protein, functional derivative thereof, or nucleic acid moleculecan be administered to the subject in any practical and convenientmanner. Suitable routes of administration include the oral, nasal,topical, transdermal, and parenteral (e.g., intravenous,intraperitoneal, intradermal, subcutaneous or intramuscular) route. Inaddition, a Nell1 protein, functional derivative thereof, or nucleicacid molecule can be introduced into the body, by injection or bysurgical implantation or attachment, proximate to a preselected tissueor organ site such that the Nell1 material is able to enter the site bydirect diffusion. For example, a Nell1 protein, functional derivativethereof, or nucleic acid can be provided in a patch or gel likesubstances, which, upon administration (by e.g., injection orimplantation) can be taken up directly by tissues as a result ofdiffusing from a site of high concentration to one where there is verylow level of the substance. If Nell1 protein, functional derivativethereof, or nucleic acid molecule is administered locally, theformulation is such that the Nell1 protein, functional derivativethereof, or nucleic acid molecule does not diffuse and adversely affectsurrounding organs.

Alternatively, a Nell 1 protein, or functional derivative thereof, canbe administered directly to injured and damaged tissue (e.g., infarctand surrounding border zones). Such administration, can be applied, forexample, to treat cardiovascular defects, thus minimizing heart muscleinjury or stimulating tissue repair processes in the heart after MI.

Other delivery systems and methods include, but are not limited to: a)catheter-based devices that permit site specific drug delivery to theheart muscle, b) via a thorascopic opening (small minimally invasivewound in the thoracic cavity; similar to laparascopic methods) throughwhich a scope and guided injection device containing Nell1 protein,derivative thereof, or nucleic acid molecule is introduced, c)ultrasonic-based drug delivery methods (see, for example, Mayer et al.,Advanced Drug Delivery Reviews, 2008, 60:1177-1192 and Bekeredjian etal., Ultrasound in Medicine and Biology, 2005, 31:687-691), and d)infusion into the pericardial space (see, for example, Xiao et al., Am.J. Physiol, Heart Circ. Physiol., 2008, 294:H12212-12218).

Important general considerations for design of delivery systems andcompositions, and for routes of administration, for protein/peptidedrugs may apply. For example, the appropriate delivery system for Nell1protein and/or functional derivatives thereof will depend upon itsparticular nature, the particular clinical application, and the site ofaction.

Formulations for oral delivery or systemic delivery, for instance, mayrequire certain considerations due to, for example, instability of Nell1protein and/or functional derivatives thereof in the gastrointestinaltract, or exposure of Nell 1 protein and/or functional derivativesthereof to proteases. Any method known to those skilled in the art canbe utilized to address such considerations.

For example, for oral delivery, an absorption-enhancing agent can beutilized. A wide variety of absorption-enhancing agents have beeninvestigated and/or applied in combination with protein compositions fororal delivery and for delivery by other routes (van Hoogdalem, Pharmac.Ther. 44, 407-43, 1989; Davis, J. Pharm. Pharmacol. 44(Suppl. 1),186-90, 1992). Most commonly, typical enhancers fall into the generalcategories of (a) chelators, such as EDTA, salicylates, and N-acylderivatives of collagen, (b) surfactants, such as lauryl sulfate andpolyoxyethylene-9-lauryl ether, (c) bile salts, such as glycholate andtaurocholate, and derivatives, such as taurodihydrofusidate, (d) fattyacids, such as oleic acid and capric acid, and their derivatives, suchas acylcarnitines, monoglycerides, and diglycerides, (e)non-surfactants, such as unsaturated cyclic ureas, (f) saponins, (g)cyclodextrins, and (h) phospholipids.

Alternatively, Nell 1 protein and/or functional derivative thereof, canbe administered in combination with other drugs or substances thatdirectly inhibit proteases and/or other potential sources of enzymaticdegradation of proteins. Yet another alternative approach to prevent ordelay gastrointestinal absorption of Nell1 protein and/or functionalderivative thereof is to incorporate them into a delivery system that isdesigned to protect the protein from contact with the proteolyticenzymes in the intestinal lumen and to release the Nell1 protein and/orfunctional derivatives thereof at the site of cardiovascular injury. Amore specific example of this strategy is the use of biodegradablemicrocapsules or microspheres, both to protect a protein fromdegradation, as well as to effect a prolonged release of active protein(see, for example, Deasy, in Microencapsulation and Related Processes,Swarbrick, ed., Marcell Dekker, Inc.: New York, 1984, pp. 1-60, 88-89,208-11).

In a specific embodiment, a Nell 1 protein, functional derivativethereof, or nucleic acid molecule is administered to directly repairheart muscle after MI. Delivery can be performed via direct delivery toor near the injured heart muscle site (infarct and border zones) byinjection, by catheter, via absorbable biomatrix (i.e. biocompatibleporous) material, and the like, and combinations thereof. According tothis embodiment, the Nell 1 composition is administered to the subjectafter the initial inflammatory responses subsides—usually within 72hours, within 48 hours, within 36 hours, within 24 hours, or even within18 hours of MI, in order to minimize the extent of the injury andachieve better therapeutic efficacy. There is a flood of inflammatoryresponses immediately after heart muscle injury. It is believed to beoptimal to administer Nell 1 after this initial defensive response ofthe surrounding tissue. Regenerative processes, which naturally beginsafter the inflammatory response slows down, are where Nell1 is likely towork best.

Further according to the present invention, a Nell1 protein, functionalderivative thereof, or Nell 1-encoding nucleic acid molecule can be usedindependently or in conjunction with additional therapeutic compositionsuseful for treating a cardiovascular condition.

In a specific embodiment, a Nell1 protein, functional derivativethereof, or Nell 1-encoding nucleic acid molecule is used together withstem cells for the repair and regeneration of damaged cardiac musclesand blood vessels.

Cell-based therapies for the repair and regeneration of damaged cardiacmuscles and blood vessels utilize implantation of cells (such ascardiomyocytes), or introduction of stem cells isolated from adult bonemarrow to develop new cardiac muscle in the area of implantation. See,e.g., Orlic et al., 2001; Rubart et al., 2006; Ott et al., 2006;Rosenthal et al., 2006. Without being bound by theory, the use of Nell1increases the efficiency of cell-based therapies for the repair andregeneration of damaged cardiac muscles and blood vessels.

According to the present invention, a Nell1 protein or nucleic acidmolecule can be co-delivered with the appropriate cells, e.g.,cardiomyocytes or adult stem cells, directly to the damaged sites of asubject using biological matrices or direct injection methods already inpractice for cell-based therapies.

In another embodiment, a Nell1 protein, functional derivative thereof,or Nell 1-encoding nucleic acid molecule is used in vitro to stimulateor promote the development and differentiation of stem cells intocardiomyocytes useful for the repair and regeneration of damaged cardiacmuscles and blood vessels. See, for instance, example 7.

This invention is further illustrated by the following examples, whichare not to be construed in any way as imposing limitations upon thescope thereof. The terms and expressions which have been employed in thepresent disclosure are used as terms of description and not oflimitation, and there is no intention in the use of such terms andexpressions of excluding any equivalents of the features shown anddescribed or portions thereof. It is to be understood that variousmodifications are considered to be included within the scope of theinvention. All the publications mentioned in the present disclosure areincorporated herein by reference.

Example 1 Nell1^(6R) Mutant Mouse

The Nell1^(6R) mutant mouse was used in the experiments described in thefollowing examples. Generation, breeding and maintenance of this mutantmouse is described in U.S. Published Application 2006/0053503, which isincorporated herein by reference. Briefly, the mutant mouse contains arecessive neonatal-lethal point mutation in the Nell1 gene, originallyinduced by N-ethyl-N-nitrosourea (ENU). Nell1^(6R) has T to A basechange that converts a codon for cysteine into a premature stop codon(TGT to TGA; Cys(502)Ter), resulting in a severe truncation of the Nell1protein product and a marked reduction in steady state levels of theNell transcript.

Example 2 Heart Defects in Nell1^(6R) Mutant Mouse

Formalin-fixed specimens were analyzed by heart length and widthmeasurements. These measurements were completed on wild type,heterozygous, and mutant mice at the 18.5-day embryonic stage. Furtherobservations were made using standard histological methods (haematoxylinand cosin staining on mouse sagittal sections).

Nell1^(6R) mice were observed to have significantly enlarged heartsbased on length and width measurements. As shown in Table 1, lengthmeasurements for all three genotypes did not differ significantly.However, based on the statistical T-test, the width measurements formutant mice was significantly greater compared to the width for wildtype and heterozygous mice, this confirming presence of an abnormalheart phenotype in mutant mice.

Examination of the haematoxylin and eosin-stained slides showeddramatically reduced blood flow out of the heart. As shown in FIGS. 1-2,wild-type and heterozygote mice showed arteries filled with bloodwhereas blood was not a very prominent feature in slides of mutant mice.Therefore, the loss of Nell 1 function resulted in a significantreduction of the number of blood vessels and extensive branching of thevasculature in mutants as compared to wild type fetuses. The decrease inblood vessel formation was observed throughout the fetal body.

In addition, a larger number of immature heart cells and lesserextracellular matrix were observed in mutant mice as compared to wildtype mice (FIG. 3A-3B). The dense packing of smaller cardiomyocytes inthe mutant (FIG. 3B) was very apparent in the denser/darker stainingwith haematoxylin and eosin, compared to the wild type (FIG. 3A).

TABLE 1 Measurements of Nell1^(6R) Hearts Indicating Heart EnlargementMeasurement (mm) of E18.5 fetal heart width and length of NelI1^(6R)heterozygote and homozygote mutant mice compared with wild-typelittermates. There is significant enlargement of fetal hearts inhomozygote mutant compared to the heterozygotes and normal mice.Homozygote Heteozygote Wild-type Nell16R/Nell 16R +/Nell 1 6R +/+ Width3.3 2.8 2.7 2.5 2.8 2.8 2.8 2.3 2.8 2.3 2.7 2.5 2.8 2.8 2.7 3.0 2.5 2.33.2 2.5 2.2 3.0 2.5 2.5 2.8 2.2 2.8 2.8 2.5 2.7 3.3 2.2 2.7 3.0 — 2.23.0 — 2.5 2.5 — 2.3 2.5 — — 3.0 — — 2.5 — — No. of Fetuses 17 11 14Average 2.853 2.530 2.476 Length 3.2 3.7 2.7 2.8 3.2 2.7 2.8 2.8 3.3 2.73.2 3.0 3.0 3.2 3.3 3.2 3.0 3.0 3.2 3.0 2.5 3.3 3.3 3.0 3.0 2.8 3.3 2.83.0 3.2 3.2 2.8 3.3 3.2 — 2.7 3.2 — 2.8 2.8 — 3.0 2.5 — — 3.0 — — 2.8 —— No. of Fetuses 17 11 14 Average 2.984 3.091 2.988 T-Test p-valuesMutant: Mutant: Heterozygote: Wild-type Heterozygote Wild-type Width0.0012442891 0.0046893426 0.6143698331 Length 0.9351530349 0.24709112300.3514701862

These above cardiovascular defects were evident in E18.5 day fetusesrecovered by caesarean. Additionally, wild type and heterozygote micehad spongy lungs that filled their entire thoracic cavity, while mutantmice had compact, dense lungs. Mutant mice did not survive birth. Theseverity of the heart and blood vessel defects were likely to be thecause of the death of the fetuses during the birth process reportedearlier (Desai et al, 2006). Fetuses that were recovered by caesareanwere unable to breathe as depicted in the collapsed lung in the mutants.

Example 3 ECM Genes Affected by Nell1 Influence Heart Development

A comprehensive gene expression analysis using public database (UCSCGenome Browser, Mouse Genome Informatics, Integrated Cartilage GeneDatabase, PubMed) was conducted to investigate the relationship betweencardiovascular development and each of the 28 extracellular matrix (ECM)genes which were shown previously (Desai et al., 2006) to exhibitreduced expression in Nell1^(6R) mutant mouse bodies. Of the 28 ECMgenes studied, the bioinformatics analysis showed that the majority ofgenes with reduced expression in Nell-1 deficient mice are normallyexpressed in the heart (79% of the analyzed ECM genes; 22/28), bloodvessels (71%; 20/28) and bone marrow (61%; 17/28) (See Table 2). TheMouse Genome Informatics database referenced several genes (Col15a1,Osf-2, Bmpr1a, Pkd1, Mfge8, Ptger4, Notch3) that have been mutated inmice and actually manifest abnormalities in cardiovascular development.

Mouse mutations in some of these genes display heart deformitiescommonly associated with heart enlargement, as shown in Table 3 below.

TABLE 2 Expression profile of genes in the Nell1 pathway and associationwith mutant mouse phenotypes. # abnormal Gene Expression bone heart #total Symbol Gene Name heart vascular blood marrow phenotype¹³ mutants¹³Tnxb tenascin 10 10 11 11 2 Prg4 proteoglycan 4 33 12 12 1 Thbs3thrombospondin 3 10 10 12 2 Col5a3 collagen 5 alpha 3 subunit Neurog2neurogenin 2 5 Col5a1 procollagen type V, alpha 1 10 10 10 10 1 1 Col6a1procollagen Type VI, aloha 1 10 16 12 10 1 Col15a1 procollagen type XV,alpha 1 10 19 10 12 1 1 Pacsin3 PKC and casein kinase 10 10 substrate inneurons 3 Tnc tenascin c 10 10 21 11 3 Col12a1 procollagen type XII,alpha 1 10 12 10 Chad chondroadherin 15 15 Osf2- osteoblast specificfactor 2 10 10 10 1 2 pending Col17a1 procollagen type XVII alpha 1Prkcc protein kinase C 2 Prkch protein kinase C, eta symbol 10 10 10 101 Bk- brain and kidney protein pending Ptk9l PTK9L protein tyrosine 1010 10 10 kinase 9-like Npdc1 neural proliferation, 10 10 1differentiation and control gene Bmpr1a bone morphogenetic protein 10 1210 2 4 receptor type 1a Pkd1 polycystic kidney disease I 10 27 10 12 712 homolog Tnfrsf11b tumor necrosis factor (ligand) 10 34 12 3 Mfge8milk fat globule-EGF factor 8 10 12 10 10 1 5 protein Matn3 matrilin 3,cartilage matrix 28 1 protein Bmp7 bone morphogenetic protein 10 10 8type 7 Matn2 matrilin 2, cartilage matrix 10 10 10 10 2 protein 2 Ptger4prostaglandin E receptor 4 10 10 10 3 4 Notch3 notch gene homolog 3 1030 4 # of Genes 22 20 13 17 7 20 Percentage 79% 71% 46% 61% 25% 71%

TABLE 3 Mutated genes causing heart defects associated with enlargementGene Defect Col6a1 Dilated descending aorta Bmprla Persistent truncusarteriosus Outflow tract formation abnormalities Pkd1 Vascularleaks/ruptures Endocardial cushion defects Abnormal atrial septummorphology Double outlet right ventricle Abnormal septation Bmp7 Lack ofendocardial cushion formation Ptger4 Dilated left ventricle Patentducrus arteriosus Congestive heart failure

Example 4 Gene Expression in Nell1^(6R) Mutant Mouse

To define the involvement of Nell1 in the known molecular pathways thatgovern heart structure and function, a comprehensive gene expressionanalysis was conducted in the entire mouse genome (30,000 genes) ofnormal fetal hearts and those dissected from Nell1^(6R). This analysisconsisted of 50 mutant fetal hearts separated into 4 pools of 10-13hearts and 35 normal hearts separated into three pools of 10-12 hearts(18.5 days of gestation). RNAs were extracted from the pooled tissues,processed for microarray analysis on the Illumina Mouse V6 chips andscanned with Illumina Beadstation 500GX. Data was analyzed with theBeadStudio software and Gene Ontology Tree machine. At least 345 geneswere identified that were differentially expressed between normal andmutant samples (at p value=0.001 for the microarray detection anddifferential p values; denotes a very high statistical significance).Table 4 lists a representative sampling of genes influenced by Nell1that already have established functions in cardiovascular conditions.Table 4 also provides the literature references for the specific studiesthat have demonstrated these gene functions.

Table 4 shows a number of genes in the Nell1 pathway that have beenimplicated in the processes that ensue after heart failure. The abilityof Nell1 to stimulate proteins that control cell differentiation andproper secretion of the cardiac ECM strongly suggests that this proteincan restore proper ECM constitution and orientation in heart muscleafter a heart attack, thereby preventing or alleviating heart muscledamage and subsequent loss of heart function (or death) resulting fromMI.

Example 5

The data presented here were based on studies of the Nell1^(6R) mutantmouse. Rodent Nell1 studies are believed to translate accurately to thehuman situation. The complete mouse Nell1 coding sequence has beenreported (Genbank Accession No. AY622226; Desai et al., 2006). Acomparison of this sequence with the most current human Nell1 gene inthe public genome databases (UCSC Genome Browser and NCBI) indicates avery high homology of 87% gene sequence identity. The corresponding810-amino acid residue polypeptides have a 93% identity in their aminoacid sequences (FIG. 5). When one considers conservative substitution ofsimilar amino acids, the human and mouse Nell1 proteins are 97%conserved. This remarkable degree of gene and protein structureconservation suggests the conservation of functions and fundamentalmechanisms of Nell1-mediated pathways in human and mouse.

Example 6 Animal Model for Assessing Therapeutic Efficacy of Neil for MI

The efficacy of the Nell 1 protein for regenerating cardiac muscle afterdamage induced by a myocardial infarction (MI) is tested in a widelyused and accepted in vivo animal model. Myocardial infarction is inducedin a murine in vivo model by blocking the main blood supply line to theleft ventricle. The surgical procedures for generating this model aredescribed in detail by several publications (Patten et al., 1998;Tarnayski et al., 2004; Ahn et al., 2004).

Briefly, mice are anesthetized, restrained in a supine position, andintubated with pure oxygen regulated by a small animal ventilator. Athoracotomy is performed under a dissecting scope, at the fourth orfifth intercostal space of the left side, between the heart and lungmargins. The thoracic surgical hole is enlarged using retractors and thepericardial sac is gently torn with fine forceps.

The left anterior descending coronary artery (LAD) is visualized andligated by passing a tapered microsurgical needle (¼ circle, 140microns) with a black silk monofilament suture (size 7 or 8) underneaththe coronary artery and tying the suture to completely stop the bloodflow in the artery. A small polyethylene tubing (PE 10) 2-3 mm is placedbetween the tie and the LD to minimize cutting and severely injuring theartery.

Myocardial infarction is confirmed by observing for blanched or whiteappearance of the left vertical that correspond to the muscles that havelost blood supply and the alteration of the wave pattern (pronounced STwave elevation) in an electrocardiogram. Since the LAD provides theblood supply to the left ventricle, this surgically-induced myocardialinfarction will cause the death of myocardial tissue (necrosis) in theleft bentricular wall and the anterior section of the interventricularsection. The size of the myocardial infarction lesions/infarcts can becontrolled by the exact position of the ligation along the LAD. Ligationat a high position (atrioventricular junction) will reduce blood flow toa larger area and make larger infarcts while ligations at lower areaswill make medium or small lesions. Ligature position is kept constantfor any given experimental group to keep the infarction size constate.

After myocardial infarction induction, the thoracic and skin wounds aresutured and mice are allowed to recover from anesthesia on a heating pador with heat lamps.

To test the ability of Nell1 to repair cardiac tissue damage due to anacute myocardial infarction event, purified Nell1 protein are delivereddirectly into the surrounding tissue around the visible infarct andwithin the infarct. Direct delivery of Nell1 protein is performed byreopening the original thoracic wound used to induce the infarct.

Nell1 and functional derivatives thereof containing EGF like domainsand/or the von Willebrand like domain of Nell1 are administered at 2-3points along one side of the infarct border zone. In some animals,direct delivery of Nell1 protein is administered via microinjection,application of Nell1 in a gel or microspray, via nanoparticles, ortime-release patches. In others, it is administered via a Nell1 proteinexpression vector (continuous delivery). Administration of Nell1 isperformed after the initial surge of inflammatory response triggered bycardiac damage and at the time heart tissue attempts innate regenerativemechanisms (approximately 4-5 hrs after MI). The effects of Nell1administration are evaluated by standard histology andimmunohistochemistry techniques for detection of proteins associatedwith cardiac tissue regeneration (Orlic et al., 2001).

Example 7 In vitro Stem Cell Therapy

A promising approach in the field of heart muscle regeneration after MIis the introduction of either embryonic or adult mesenchymal stem cellsinto the damaged heart. However, data indicate that although new heartmuscle cells can be regenerated that the new tissue may not necessarilydisplay the full functional capacity of mature heart tissue(contractility).

To promote full functional capacity of mature heart tissue, Nell 1protein and functional derivatives thereof containing EGF like domainsand/or the von Willebrand like domain of Nell1 are co-delivered withstem cells to the injured heart muscle using the same strategiescurrently in use for stem cell delivery.

Example 8 Animal Model for Assessing Therapeutic Efficacy of Nell1 forMyocardial Ischemia and Reperfusion Injury

The efficacy of the Nell1 for regenerating cardiac muscle after damageinduced by myocardial ischemia and reperfusion injury is tested in awidely used and accepted in vivo animal model. Myocardial ischemia andreperfusion injury is induced in an in vivo murine model as follow:

-   1. After anesthesia, intubation and hook-up to a mouse ECG machine,    the chest cavity of the mouse is opened at the intercostal space    (usually 4^(th) or 5^(th)) and the opening is retracted to reveal    the left side of the heart and to locate the LAD artery. The    pericardial sac is torn gently with forceps and the LAD is    positioned for easy access. All surgical steps are done under a    dissecting microscope.-   2. A tapered needle (¼ circle 140 microns) with a size 8 silk or    monofilament suture is partially passed underneath the artery. A    small tubing 1-1.5″ in length (e.g. polyethylene size 10 tubing) is    placed on top and parallel to the LAD artery and perpendicular to    the length of the needle. The suture is then pulled and a surgical    tie is made such that the tubing is tied with the artery located    beneath it.-   3. The interruption of blood flow to the left ventricular heart    muscles is easily visualized by a blanched or white appearance of    the affected region (where infarct develops). The ECG will confirm    the ischemia by the alteration of the wave pattern (e.g. ST segment    elevation, T wave anomalies) compared to the normal pattern. The    change indicates that the LAD is successfully ligated and restricted    blood flow to the left ventricle has functionally induced an    ischemic event.-   4. The chest cavity and the skin are sutured such that one end of    the tubing is sticking out of the thoracic area above the sutured    skin. After the desired amount of time of ischemia, the tubing is    gently pulled out to relax the knot/ligated suture thereby allowing    reperfusion of blood into the affected area.-   5. Reperfusion is indicated by the return of the ECG pattern to    normal or near normal pattern. Different groups of mice with varying    times of occlusion before reperfusion are made.-   6. Varying concentrations of Nell1 protein are administered via    intraperitoneal injection or using a catheter device that is placed    before the chest cavity is closed after LAD ligation and ischemia.    The catheter device allows for controlled delivery so that Nell1    protein can be delivered immediately after reperfusion or given time    points after reperfusion is induced. In other models, Nell1 protein    is administered by reopening the surgical sutures and re-entry to    the chest cavity and direct Nell1 delivery by microinjection or gel    patch.

Example 9 Animal Model for Assessing Therapeutic Efficacy of Neil forCardiac Hypertrophy

The use of Nell1 protein as a therapeutic for cardiac hypertrophy istested in a widely used and accepted in vivo animal model. Cardiachypertrophy is generated by physical/surgical means [pressure-overload].

In the in vivo pressure overload animal model, the aorta of a mouse/rator large animal is banded to reduce the diameter and thus the blood inthe left ventricle builds up pressure and induces hypertrophy of theleft ventricle (Tarnayski et al 2004). This type of animal model mimicsthe human condition of aortic stenosis where the narrowing of the aorticvalve restricts blood flow from the left ventricle to the aorta. Thepersistent increased pressure in the left ventricle leads to increase inmuscle mass (hypertrophy) of the walls. This model is generated asfollows:

-   -   1. Mice are anesthesized and a 5 mm transverse incision is made        at the level of the left armpit, 2 mm away from the sternal        border. A small incision (5 mm) is made at the 2^(nd)        intercostal space and opened with microretractors.    -   2. The thymus and fat covering the aortic area are pushed away        and the pericardial sac is gently torn. The ascending portion of        the aorta is located and bluntly dissected from the pulmonary        trunk and forceps is placed underneath the ascending aorta    -   3. A 7-0 silk suture is placed around the aorta and a loose knot        is made. A 25 or 27 gauge needle (outer diameter of 0.51 mm)        that is bent into an L shape is placed through the loose loop,        positioned above and parallel to the aorta and a second knot is        tied securely. The needle is retracted to yield a constricted        aorta (60-80% constriction for a 27 gauge). Two more knots are        tied.    -   4. The chest cavity is closed by suturing ribs and then the skin        wound.

Nell1 protein and functional derivatives thereof containing EGF likedomains and/or the von Willebrand like domain of Nell 1 are administeredas an injectable after the onset of hypertrophic changes and heartfunction anomalies detected by ECG. Times of administration are testedas one high dose after hypertrophy is diagnosed or at lower doses givenmultiple times (weekly) after hypertrophy is diagnosed. Efficacy of thetreatment is evaluated by quantitative measurements of ventricular andheart size, physiological monitoring by ECG and other heartvisualization tools, molecular markers for heart failure etc. asdescribed earlier.

Example 10 Animal Model for Assessing Therapeutic Efficacy of Nell1 forCardiomyopathy

The use of Nell1 protein as a therapeutic for cardiomyopathy is testedin a widely used and accepted in vivo animal model. The in vivo mousemodel of cardiomyopathy is generated by gene-targeted approaches such asknock-outs or over-expression of a single gene, wherein the homozygotes(two mutant gene copies) and/or heterozygotes (one mutant copy) cansurvive to the juvenile or adult stage. Suitable in vivo mouse models ofcardiomyopathy contain knock-outs or over-expression of genes andpathways (e.g., (extracellular matrix and matricellular proteins,tenascins, thrombospondins, matrilins, etc.) that are controlled by theNell1 signaling protein. A specific example of an appropriate smallanimal model is the targeted knockout of the mouse Nov (Ccn3) genereported by Heath et al. (BMC Developmental Biology 2008:8:18).

Briefly, Nov (Ccn3) mutant mice are generated. Imaging of hearts byechocardiograms and electrocardiograms are conducted to determine heartfunction and presence of visible heart structure anomalies prior totreatment.

Nell1 protein and functional derivatives thereof containing EGF likedomains and/or the von Willebrand like domain of Nell1 are administeredby intraperitoneal injection to young Nov (Ccn3) mutant mice andcorresponding controls during the first two months of life. Variousdosages and timing regimens are tested. After treatment, heart functionparameters are measured in Nell1-treated and controls during the timethat untreated mutant mice show the severe symptoms of cardiomyopathy,generally at 4-5 months in Nov mice.

After cardiovascular functional/physiological studies, the mice aresacrificed and hearts are dissected and fixed for morphological andhistological evaluation such as: total heart size, chamber sizes(especially left ventricle), heart valve structure, chordae tendinae,interventricular septum, heart muscle cell (cardiomyocyte) size andappearance, vessels going in and out of the heart etc.

Example 11 NELL1 Protein Treatment of Heart Muscle Damage fromMyocardial Infarction

The ability of Nell1 protein to trigger cellular pathway(s) forregeneration of damaged heart muscle was demonstrated in an in vivomouse model. A heart attack or myocardial infarction was generated in4-5 month old adult mice (strain C57B1/6J) by surgically tying the leftanterior descending (LAD) coronary artery, which is the main bloodsupply line to the left ventricle (lv) and the interventricular septum(IVS). The left ventricle pumps oxygenated blood through the aorta intothe rest of the body while the IVS divides the right and left ventriclesof the heart. LAD ligation in animal models results in the damage andsubsequent death of the heart muscle tissue. Table 5 summarizes theresults of treating mouse hearts with the purified human NELL1 proteinon the third day post-MI event. The NELL1 protein was diluted inphosphate buffered saline (PBS) and was delivered directly onto thedamaged heart muscle as a very concentrated microdrop, while the micewere under anaesthesia and intubation for about an hour. Three mice weretreated with 312 ng and four mice with 624 ng purified NELL1 protein.Four mice underwent the same cardiac surgery but were given a microdropof PBS on the damaged heart tissue and served as controls. In additionto these controls, over 20 MI mice were previously generated and studiedto obtain consistency in MI surgical and post-surgical techniques. Theseearlier “controls” displayed the same characteristics as controlsrepresented in Table 5. All treated and untreated mice were maintainedfor an additional 14 days before they were sacrificed to collect heartsand other major organs (a total of 17 days post-MI). Heart sizemeasurements indicated slight increases in both heart width and depth inNell1-treated hearts. Remarkably ALL treated mice showed dramaticallylesser visible areas of the infarcted tissue on the surface of theheart. In 6 out of 7 hearts the damaged tissue was only visible underthe microscope after they were fixed in buffered formalin. FIG. 6A-6Dshow the range of improvement observed in NELL1-treated hearts, frombarely visible to about 30% infarct sizes in comparison to the usual50-90% infarct sizes seen in controls. FIG. 7A-7D present histologicalanalysis of sectioned hearts stained with Masson-Trichome and furtherconfirmed that there is decreased damage at the cellular level in theNELL1-treated hearts compared to the controls. At 17 days post-MI, heartmuscle tissue is severely damaged such that huge gaps appear within theuntreated heart muscle in the left ventricle to the interventricularseptum. In contrast, there is a consistent and dramatic reduction in theamount of breakdown or damage observed in the heart muscle of treatedmice. These data from an in vivo MI mouse model illustrates thatclinical approaches that will enable delivery of Nell 1 protein directlyonto damaged heart muscle will be effective in reducing the effects ofan MI event.

TABLE 4 GENES IN NELL1 PATHWAY ASSOCIATED WITH KNOWN CARDIOVASCULARDISORDERS UP (↑) OR DOWN (↓) REGULATION ASSOCIATION WITH HEART DISORDERSAND GENE and DESCRIPTION [p value ≦ 0.001] DISEASES REFERENCES Tpm2;tropomyosin 2, beta ↑4.3 Cardiac-specific myofibrillogenesis;Cardiomyopathy Denz et al., 2004 Dmn; desmuslin transcript ↑9.4Hypertrophic Cardiomyopathy; heart failure Mizuno et al., 2001 variant 1Acta1; skeletal muscle actin ↑2.8 Hypertrophic cardiomyopathy; heartfailure Lim et al., 2001 alpha 1 Tpm1 tropomyosin alpha 1 ↑4.8Hypertrophic cardiomyopathy; heart failure Wernicke et al., 2007; Kostinet al., 2007 Lgals3; lectin, ↑2.6 Acute heart failure biomarker;excellent predictor of mortality Van Kimmenade et al., Galactosebinding, soluble 3 within 60 days; increases in failure pronehypertrophied 2006; Sharma et al., hearts; aortic stenosis; inducescardiac fibroblast proliferation, 2004 collagen deposition Spp1 ↑2.3Heart contractility via control of ECM proteins Okamoto, 2007 Secretedphosphoprotein 1 Inflammation control in hypertrophy, myocardialinfarction Singh et al., 2007 (osteopontin) and heart failure, valvularstenosis Fhl1 ↑1.3 Atrial fibrillation in cardiac arrhythmia; Chen etal., 2007 Four and a half limb domains β-adrenergic inducedcardiomypathy and heart failure (β- Lim et al., 2001 blocker pathway);cardiac remodeling by transcriptional regulation and myofilamentassembly Aqp1; aquaporin 1 ↑1.3 Myocardial edema Egan et al., 2006 ll6st↑1.5 Cardiac hypertrophy Terrell et al., 2006 Interleukin 6 signal Coleset al., 2007 transducer Tnc ↓1.5 Inflammation induced tissue remodelingin acute myocardial Terasaki et al., 2007 Tenascin c infarction, acutemyocarditis and cardiomyopathy, left ventricular remodeling Tnxb ↓1.8Cardiac nerve sprouting after MI contributing to arrhythmia Lai et al.,2000 Tenascin xb and sudden cardiac death Igftbp5 ↓1.3 Atrophy; Adaptivecardiac hypertrophy Baurand et al., 2007 Insulin growth factor bindingprotein 5 Fgl2 ↓1.4 Acute congestive heart failure without structural Muet al., 2007 Fibrinogen-like protein abnormalities; contractiledysfunction and rhythm abnormalities Ctgf; connective tissue ↓1.3Excessive myocardial fibrosis and diastolic heart failure Koitabashi etal., 2007 growth factor Dpt; dermatopontin ↓1.5 ECM remodeling inmyocardial infarction Takemoto et al., 2002 Ldlr; low densitylipoprotein ↓1.5 Heart failure Weiss et al., 2006 receptor Nppb ↓1.3Cardiac fibrosis Tamura et al., 2000 Natriuretic peptide precursorCongestive heart failure and myocardial infarction Hejmdal et al., 2007type b Biomarker for heart failure Seferian et al., 2007 Doust et al.,2004 Nppa ↓1.5 Cardiac fibrosis Tamura et al., 2000 Natriuretic peptideprecursor Congestive heart failure and myocardial infarction Hejmdal etal., 2007 type a Biomarker for heart failure Seferian et al., 2007 Doustet al., 2004 Ttn ↓1.4 Cardiac muscle dystrophies (contractility)Fougerousse et al., 1998; Titin Koatin et al., 2000 Cyr61 ↓1.7Inflammatory cardiomyopathy Wittchen et al., 2007; Cysteine rich protein61 Mo and Lau, 2006 Sgcb Cardiac muscle dystrophies Fougerousse et al.,1998 Sarcoglycan

TABLE 5 Results of Nell1 Protein Treatment of Damaged Heart Tissue in aMouse Model with Myocardial Infarction Heart length Heart Width HeartDepth Estimated Infarct Size Mouse Weight Change Top-Bottom Left-RightFront-Back 17 days post-MI Number 17 day period (mm) (mm) (mm) (% leftventricle) Controls (PBS) m2589 0 8.32 5.84 4.91  75% m2588 +1.2 8.556.20 5.11  50% m2733 −0.9 8.78 7.01 5.69  60-70% m2764 +1.1 8.52 6.095.57  90% Average +0.35 8.54 6.28 5.32 ~70% Nell1 Protein Dose I (312ng) m2550 −3.2 8.42 7.41 6.19 Infarct hardly visible until fixation;~16% faint area m2597 −2.3 8.04 6.12 6.17 Infarct barely visible untilfixation; 30% faint area m2553 −2.3 9.21 6.44 5.52 Infarct hardlyvisible until fixation; 30% faint area Average −2.6 8.56 6.66 5.96~25.3% Nell1 Protein Dose II (624 ng) m2668 +0.1 8.51 6.55 5.65 Infarcthardly visible until fixation; 25% faint area m2732 −0.1 8.94 6.44 5.73Infarct hardly visible until fixation; 10% very small faint area m2726−2.7 8.50 6.90 5.94 Infarct hardly visible until fixation; very faintlayer difficult to estimate ra2727 −0.3 8.42 6.95 6.26 Visible infarctat ~30% Average −0.75 8.59 6.71 5.90 ~16.3%

REFERENCES

-   Aghaloo T et al. Am. J. of Path 2006; 169:903-915.-   Ahn D et al. Am J Physiol Heart Circ Physiol 2004, 286:1201-1207.-   Baurand A et al, Circ Res 2007; 100:1353-1362.-   Chen C L et al. Biochim Biophys Acta 2007; 1772: 317-329.-   Coles B et al. Am J Pathol 2007; May 3 Epub.-   Cundy T et al. Hum Mol Genet 2002; 11:2119-2127.-   Denz C R et al. Biochem Biophys Res Commun 2004; 320:1291-1297.-   Desai J et al. Hum Mol Genet 2006; 15:1329-1341.-   Doust J A et al. Arch Intern Med 2004; 164: 1978-1984.-   Egan J R et al. Biochim Biophys Acta 2006; 1758:1043-1052.-   Fougerousse F et al. Genomics 1998; 48:145-156.-   Grahame R et al. Ann Rheum Dis 1981; 40:541-546.-   Helmjdal A et al. J Card Fail 2007; 13:184-188.-   Jackson G C et al. J Med Genet 2005; 41:52-59.-   Kostin S et al. Heart Fail Rev. 200 5:271-280.-   Koitabashi N et al. Hypertension 2007; 49:1120-1127.-   Kuroda et al., Biochemical Biophysical Research Comm. 265: 79-86    (1999a).-   Kuroda et al., Biochemical Biophysical Research Comm. 265: 752-757    (1999b).-   Lai A C, et al. J Cardiovasc Electrophysiol 2000; 11:1345-1351.-   Leier C V et al. Ann Intern Med 1980, 92:171-178.-   Lim D S et al. J Am Coll Cardiol 2001; 38:1175-1180-   Liu L et al. Journal of Undergraduate Research (Vol. 7). 2007.-   Lu et al. The Spine Journal 2007; 7: 50-60.-   Mao J R et al. J Clin Invest 2001; 107: 1063-1069.-   Mao J R et al. Nat Genet 2002; 30:421-425.-   Mizuno T et al. BMC Genet 2001; 2: 8.-   Mizuno Y et al. Proc Natl Acad Sci U.S.A. 2001; 98: 6156-6161.-   Mo F E et al. Circ. Res. 2006; 99: 961-969.-   Mu J et al. Physiol Genomics Jun. 5, 2007 (Epub).-   Okamoto H. Mol Cell Biochem 2007; 300:1-7.-   Orlic D et al. Ann N Y Acad Sci 2001; 938:221-229.-   Orlic D et al. Nature 2001; 410: 701-705.-   Ott et al. Expert Opin Biol Ther 2006; 6(9): 867-78.-   Patten R D et al. Am J Physiol Heart Circ Physiol. 1998;    274:1812-1820.-   Rosenthal et al., Cell Transplant 2006; 15 Suppl 1: S41-5.-   Rubart et al., Ann NY Acad Sci 2006; 1080: 34-48-   Seferian K R et al. Clin Chem 2007; 53:866-873.-   Sharma U C et al. Circulation 2004; 110:3121-3128.-   Singh M et al. Front Biosci 2007; 12:214-221.-   Stem cell repair in ischemic heart disease: an experimental model.    Int J Hematol. 2002; 76 Suppl 1:144-145.-   Sussman, Nature 2001; 410: 640-641.-   Takemoto S et al. Basic Res Cardiol 2002; 97: 461-468.-   Tamura et al. Proc Natl Acad Sci USA 2000; 97:4239-4244.-   Tarnayski O et al. Physiol Genomics 2004; 16:349-360.-   Terrell et al. Shock 2006; 26:226-234.-   Terasaki F et al. Circ J 2007; 71:327-330.-   Ting K. et al. J of Bone and Mineral Research 1999; 14:80-88.-   van Kimmenade et al. J. Am. Coll. Cardiol. 2006; 48: 1217-1224-   Weiss R M et al. Circulation 2006; 114: 2065-2069.-   Wernicke D et al. Biomed Tech (Berl) 2007; 52: 50-55-   Wittchen F et al. J Mol Med 2007; 85:253-267.-   Zhang X et al. J Bone Miner Res 2003; 18:2126-2134.-   Zhang X et al. J Clin Invest 2002; 110:861-870.

What is claimed is:
 1. A method of treating a cardiovascular disorder ina subject in need thereof comprising administering a Nell1 protein tosaid subject.
 2. The method of claim 1, wherein said Nell1 proteincomprises an amino acid sequence as set forth in any one of SEQ ID NO:2, SEQ ID NO: 4 or SEQ ID NO:
 6. 3. A method of treating acardiovascular disorder in a subject in need thereof comprisingadministering a nucleic acid coding for a Nell1 protein to said subject.4. The method of claim 3, wherein said nucleic acid is an expressionvector to effect expression of Nell1 in said subject.
 5. The method ofclaim 4, wherein said expression vector is a viral or non-viral vector.6. The method of any one of claims 1-5, wherein said cardiovasculardisorder is myocardial infarction, heart failure, cardiac ischemia,hypertrophy, or cardiomyopathy.
 7. The method of claim 6, wherein saidNell1 protein or said nucleic acid is administered systemically.
 8. Themethod of claim 7, wherein said Nell1 protein or said nucleic acid isadministered by ingestion, injection or implantation.
 9. The method ofclaim 6, wherein said Nell1 protein or said nucleic acid is administeredlocally.
 10. The method of claim 9, wherein said Nell1 protein or saidnucleic acid is administered by injection or implantation at or near thesite of cardiac muscle damage.
 11. The method of claim 6, wherein saidNell1 protein or said nucleic acid is administered via catheter to ornear the site of cardiac muscle damage.
 12. The method of claim 6,wherein said Nell1 protein or said nucleic acid is administered inconjunction with cells for the repair and regeneration of damagedcardiac muscles and blood vessels.
 13. The method of claim 12, whereinsaid cells are cardiomyocytes.
 14. The method of claim 12, wherein saidcells are stem cells.
 15. A method of treating myocardial infarction ina subject in need thereof comprising administering a Nell1 protein tosaid subject.
 16. A method of treating heart failure in a subject inneed thereof comprising administering a Nell1 protein to said subject.17. A method of treating cardiac ischemia in a subject in need thereofcomprising administering a Nell1 protein to said subject.
 18. A methodof treating hypertrophy in a subject in need thereof comprisingadministering a Nell1 protein to said subject.
 19. A method ofcardiomyopathy in a subject in need thereof comprising administering aNell1 protein to said subject.
 20. The method of anyone of claims 15-19,wherein said Nell1 protein is administered in conjunction with cells.21. The method of claim 20, wherein the cells are cardiomyocytes. 22.The method of claim 20, wherein the cells are stem cells.